Spalding Tracy A, Ma Jian-Nong, Ott Thomas R, Friberg Mikael, Bajpai Abhishek, Bradley Stefania Risso, Davis Robert E, Brann Mark R, Burstein Ethan S
ACADIA Pharmaceuticals, Inc., 3911 Sorrento Valley Boulevard, San Diego, CA 92121, USA.
Mol Pharmacol. 2006 Dec;70(6):1974-83. doi: 10.1124/mol.106.024901. Epub 2006 Sep 7.
Transmembrane domain 3 (TM3) plays a crucial role mediating muscarinic acetylcholine receptor activation by acetylcholine, carbachol, and other muscarinic agonists. We compared the effects of point mutations throughout TM3 on the interactions of carbachol, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), a potent structural analog of AC-42 called 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), N-desmethylclozapine, and clozapine with the M(1) muscarinic receptor. The binding and activation profiles of these ligands fell into three distinct patterns; one exemplified by orthosteric compounds like carbachol, another by structural analogs of AC-42, and a third by structural analogs of N-desmethylclozapine. All mutations tested severely reduced carbachol binding and activation of M(1). In contrast, the agonist actions of AC-42 and AC-260584 were greatly potentiated by the W101A mutation, slightly reduced by Y106A, and slightly increased by S109A. Clozapine and N-desmethylclozapine displayed substantially increased maximum responses at the Y106A and W101A mutants, slightly lower activity at S109A, but no substantial changes in potency. At L102A and N110A, agonist responses to AC-42, AC-260584, clozapine, and N-desmethylclozapine were all substantially reduced, but usually less than carbachol. D105A showed no functional responses to all ligands. Displacement and dissociation rate experiments demonstrated clear allosteric properties of AC-42 and AC-260584 but not for N-desmethylclozapine and clozapine, indicating that they may contact different residues than carbachol to activate M(1) but occupy substantially overlapping spaces, in contrast to AC-42 and AC-260584, which occupy separable spaces. These results show that M(1) receptors can be activated in at least three distinct ways and that there is no requirement for potent muscarinic agonists to mimic acetylcholine interactions with TM3.
跨膜结构域3（TM3）在介导毒蕈碱型乙酰胆碱受体被乙酰胆碱、卡巴胆碱及其他毒蕈碱激动剂激活的过程中发挥着关键作用。我们比较了TM3上各个点突变对卡巴胆碱、4-正丁基-1-[4-(2-甲基苯基)-4-氧代-1-丁基]哌啶盐酸盐（AC-42）、一种名为4-[3-(4-丁基哌啶-1-基)-丙基]-7-氟-4H-苯并[1,4]恶嗪-3-酮（AC-260584）的AC-42强效结构类似物、N-去甲基氯氮平以及氯氮平与M(1)毒蕈碱受体相互作用的影响。这些配体的结合和激活谱分为三种不同模式；一种以卡巴胆碱等正构化合物为代表，另一种以AC-42的结构类似物为代表，第三种以N-去甲基氯氮平的结构类似物为代表。所有测试的突变都严重降低了卡巴胆碱与M(1)的结合及激活。相比之下，W101A突变极大地增强了AC-42和AC-260584的激动剂作用，Y106A使其稍有降低，S109A使其稍有增加。氯氮平和N-去甲基氯氮平在Y106A和W101A突变体处显示出最大反应大幅增加，在S109A处活性略低，但效价无实质性变化。在L102A和N110A处，对AC-42、AC-260584、氯氮平和N-去甲基氯氮平的激动剂反应均大幅降低，但通常低于卡巴胆碱。D105A对所有配体均无功能反应。置换和解离速率实验表明AC-42和AC-260584具有明显的变构特性，而N-去甲基氯氮平和氯氮平则没有，这表明它们与卡巴胆碱激活M(1)时可能接触不同的残基，但占据基本重叠的空间，这与占据可分离空间的AC-42和AC-260584不同。这些结果表明，M(1)受体至少可以通过三种不同方式被激活，并且强效毒蕈碱激动剂无需模拟乙酰胆碱与TM3的相互作用。
